Subtilisin BPN is a 275 amino acid, serine protease secreted from the soil bacterium Bacillus amyloliquefaciens. It is an unusual, but not unique, example of a protein for which the native state is difficult to access from the unfolded state. The biosynthesis of subtilisin is dependent on a 77 amino acid, N- terminal prodomain, which is auto-processed to create the mature form of the enzyme. Once processed, the native conformation of mature subtilisin is difficult to reach from the unfolded state. The folding reaction can be catalyzed in vitro, however, by the addition of the 77 amino acid prodomain as a separate polypeptide. The long term goals of this proposal are: 1) To understand the kinetic barrier to subtilisin folding. 2) To understand the role of the prodomain in reducing the barrier. 3) To elucidate general relationships between thermodynamic stability, kinetic stability and facile folding. To accomplish this we propose to use rapid kinetic methods coupled with 2D NMR experiments to characterize the folding of each residue in the core structure of subtilisin in terms of thermodynamic stability (as defined by an equilibrium constant for folding) and kinetic stability (as defined by an unfolding rate). After residue specific energetic parameters are defined we will use this information to design in vitro evolution experiments to select for facile folding vs. kinetically stable mutants. By determining the folding energetics of a protein with high kinetic stability and the role of the prodomain in reducing the kinetic barrier we hope to shed light on the following questions: 1) Does the stability of intermediates correlate with folding rate? 2) Is high kinetic stability gained at the expense of the stability of intermediates? 3) Does stabilizing folding intermediates tend to occur at the expense of the stability of the whole? 4) Do mutations which accelerate folding mimic the effects of prodomain binding? 5) Do proteins which evolve in the presence of a folding catalyst tend to become more kinetically stable but less thermodynamically stable? Inefficient in vitro folding is a limiting factor in the production of many recombinant proteins of biomedical and biotechnological interest. Insight into the nature of the energetic barriers to folding and precise understanding of the mechanism of folding catalysis should lead eventually to the design of novel protein-specific foldases. Understanding the relationships between facile folding and stability should advance the fields of protein engineering, protein structure prediction and de novo protein design.